...expression observed with wild type CoChR, soCoChR localizes primarily to the cell body with very little signal...engineered microbes, used coffee grounds could be economically repurposed as starting material for the bioproduction... FLINC biosensors for live-cell imaging of biochemical activity Article contributed by Angela Holmes... live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence ...peak of 519 nm. These proteins have improved photophysical properties and can be used in quenching, FRET...

...can contain different neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin... endogenous synaptic proteins allowing them to localize to the synapse and minimize extraneous fluorescence...Jerome Goudeau, Cynthia Kenyon, and colleagues at Calico Life Sciences. Like many split FPs, split-wrmScarlet... genes whose protein products have distinct localizations. Figure 2: Split-wrmScarlet labeling ...AAV5. Find the AAV prep here The red-shifted calcium sensor jRGECO1a is now available as AAV-PHPeb. ...

... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ... the eighth generation of adenine base editors, called ABE8 (Gaudelli et al., 2020). The generated ABE8s...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...even in adult patients. In order to analyze the clinical use of ABE8 for primary human cell editing, the...

...past decade, primary cells remain difficult to genetically edit due to toxicity from electroporation or ...some cases) and minimal toxicity, including in clinically-relevant lines such as CAR-T cells. Plus, this...Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess Cas9...microscopy in HeLa cells expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from...versions. Thanks to Christophe Leterrier, whose FocalPlanes piece on mStayGold inspired and informed this...

...methyltransferase DNMT3A allows researchers to easily add biochemical marks to genetic regions from which where they...eventually, develop treatments, we need tools to specifically edit methylation sites.  Step aside, transcriptionally...interact with transcriptional machinery to modify local gene expression, confounding any potential conclusions... DNA. When dCas9 is bound to a target CpG, it physically blocks DNMT1 from that site. So, when DNMT1 methylates...Szyf 2021.   Let me leave you with a few practical considerations: Design your gRNA to overlap the...

... staying on top of the ever expanding body of biomedical literature! PubMed PubMed is the classic search...interest.  Best uses Accessing the vast body of biomedical research papers. Twitter Scientists often use...papers, and more. Saving papers There isn’t technically a way to save papers on Twitter, but you can ... contain a short summary of the topic and a chronological list of highly cited papers that contain your...also just follow feeds curated by Meta. Feeds automatically update as papers are published, similar to Facebook...

...using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment (Figure 1...recombination event then excises the DNA between the identical loxP or lox511 sites (Figure 1, bottom). Since...presence of Cre recombinase. This plasmid now specifically drives expression of mCherry instead of BFP.... rescue with in the antisense orientation (let’s call the gene “GeneR”). The key to this experiment is...information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O, Chambon P, and Ghyselinck NB. A ...

...workplace, and pursue leadership opportunities at the local, institutional, regional, and national levels. These... first at Brigham and Women’s Hospital/Harvard Medical School and then Boston University (institutions...issues faced by postdocs and institutions are not local but are national issues. I applied and was successfully...other people mostly online and on monthly phone calls. This has its challenges as meeting in person is...Chair then stepped down, I became interim Chair automatically as it was an understood expectation of any Vice-Chair...

...and solutions to research problems. This movement calls itself the Open Science movement and is part of ...and the reagents are too expensive to make it practical - but we have members looking at how to re-use...don’t throw old equipment away until you ask your local makerspace if they want to hack new life into it...somewhere near you. The place to start looking for your local space is the Hackerspace website. Markerspaces differ...fun with technology, so feel free to contact your local space and get involved! Derek Jacoby is a PhD ...

...different questions than those on the prep calls, but often the prep calls provide good ideas for interesting ...possibly with an organizer) should set up a 30 minute call with each panelist to get to know their style and...better at remembering these things on a relaxed phone call than when first confronted on a stage in front of...long introductions. The moderator asks questions, calling on 1-2 panelist for each question in a way that...

... more than 10 modules often rely on two-step hierarchical approaches using different Type IIS restriction...stability, and buffer compatibility have placed practical limits on single- or two-step assemblies. We have... While T4 DNA Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers... plate Fidelity of assembly (percent correct) Calculated colony totals Per 2 μl assembly reaction Per...outgrowth volumes described in Figure 3, with calculated yields from entire outgrowth built from 2 µl ...

...BV510 and AmCyan have almost identical emission spectra but may be called BV510 on one cytometer and AmCyan...for one another. This overlap in spectra is often called "spillover." For example, if your flow cytometer...shift towards the X axis to become a perpendicular vertical line.     In general, most spillover between samples...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...the right fit for you without the financial and logistical stress that accompanies traveling for campus ...Visits After receiving applications, universities typically invite a select group of students to continue ...and each other. Before COVID-19, financial and logistical obstacles could make touring potential programs...in-person interviews for a variety of reasons (logistical, financial, etc…) now have options to fully engage...format of interview from a travel day to an online call in response to challenges on their end. Though the...

...Preger et al., 2020. Preger et al., Journal of Biological Chemistry 2020. https://doi.org/10.1074/jbc.RA120.01289...research, which required protein compartments to co-localize multiple enzymes in a reaction pathway, the MPyV...expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use AAV ...vectors expressing GCaMP6f with this promoter. Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f...terminal providing better signal to noise for imaging calcium dynamics in fine structures. We have expanded our...

...introducing the delivery method in 2014, Manuel Goncalves lab report that AdV-mediated transduction of gRNA...Gonçalves and his colleagues at Leiden University Medical Center are now available at Addgene along with ...and very efficient introduction of DNA into therapeutically relevant, non-transformed mammalian cells. ...and used AdV vectors to edit genes in several biological systems. It has been used for instance in inherited...Viral vectors for gene therapy: Current state and clinical perspectives. Biochemistry Moscow 81:700–708 ....

... molecular biology-grade agarose containing physiological concentrations of salts and glucose. If we circled...fluorescent protein), fluorescence microscopy can theoretically be used to select specific wells from the 48... terms of growth rates, morphology or other physiological properties. An advantage of this technique is...properties, either initially, or using secondary biological screens. For example, for cancer researchers,...Neurobiology and Anatomy at the University of Maryland Medical School in Baltimore. She is particularly interested...

...in-house protocol. DNA yields for this procedure are typically in excess of 100 ng/ul, more than enough DNA to...be viewed more as a guideline, since they are typically based on the copy number of the empty plasmid ...improved plasmid DNA yield. Culture conditions play a critical role in plasmid DNA yield. Different plasmids ...are generally, but not perfectly, correlated. Typical growth times for high copy plasmids in standard...miniprepping at various time points. Measuring the optical density of the culture can indicate the number ...

... of these techniques have in common? They all typically require secondary antibodies, frequently of the...are derived from an unusual type of IgG antibody called a heavy chain antibody (HCab), which are unique...different species, nanobodies are useful for colocalization labeling of up to 3 targets (Pleiner et al ...desired fluorophores. In figure 4B below, triple colocalization is possible even when staining cells with three...multi-color staining workflow yields similar localization patterns as cells stained for one target (compare...

... science teachers at our school put out the call to local parents who would feel comfortable speaking ...parents who can answer some of the surprisingly technical questions elementary students can ask. It doesn... dangers of people using the technology in an unethical manner, but I also tried not to be an alarmist...fields in biology, are moving so quickly that it’s basically impossible for textbooks to keep up. Even the ...

...Ample distribution channels both digitally and physically. Easy to incorporate visuals if needed. Traditional...means of scicomm. Digital or print distribution.  Typically limited to a single page of information. Primarily...levels and interaction. Can bring in things to be physically interacted with. Public speaking is a common ...internet access. High probability of real-time technical difficulties Exhibits Medium to high Audience...exhibit). No remote access; limited occupancy. Typically needs to be staffed. Mixed Media Medium to...